Guide to Ion-Exchange Chromatography

Introduction Ion exchange chromatography is the reversible adsorption of charged molecules to immobilized ion groups on a matrix of an opposite charge. Separation can be selectively achieved by adsorption and release of samples from the matrix. Ion exchange starts with the equilibration of the exchanger using pH, and ionic strength. During equilibration the exchangable groups are associated with counter ions. Once equilibrium is reached and the sample added the molecules undergo addition and adsorption with an appropriate charge displace the counter ions and bind reversibly to the matrix. The...

Introduction (cont.) Types of Exchangers (cont.) Two strong exchangers are Q-Sepharose Fast Flow and SP-Sepharose Fast Flow. The charged group of Q-Sepharose is a quarternary amine which carries a nontitratable positive charge. This matrix can be used at alkaline pH values at which the positive charge of the DEAE group would have been titrated. The charged group of S-Sepharose is the sulphonyl group (-SO3¯). Method The fractionation of proteins by ion-exchange chromatography depends upon differences in the charge of different proteins. The charge of a protein depends upon the number and type...

Introduction (cont.) Method (cont.) After choice of the appropriate resin, it is mixed with buffer to form a slurry which is poured into a suitable chromatography column. The pH of this starting buffer is crucial since it will determine the charge on the proteins to be separated. The starting buffer pH should be at least one pH unit above or below the pI of the protein to be bound to the resin to ensure adequate binding. However, bear in mind that CM-cellulose and DEAE-cellulose are examples of weak ion exchangers. A weak ion exchanger is one which is ionized over only a limited pH range. Thus...

Protocol Samples The SpinColumns are supplied dry and need to be rehydrated, the bed of ionexchange resin with starting buffer allow 10-15 minutes for rehydration. After rehydration add a 2ml collection tube to the bottom of the SpinColumn and centrifuge for 1 minutes at 1000rpm. After centrifugation the protein mixture is applied. Proteins which are oppositely charged to the media at the starting pH will bind to it, so displacing the counter ions. Proteins with the same charge as the resin or with no net charge will not bind and flow straight through the column. The different proteins bound...

Factors Effecting Selectivity Protein Ionization Factors determining the pH value for elution: 1. When a protein is in a solution with a pH value above its pI its net charge is negative and binding charge will be positive anion exchange. 2. When a protein is in a solution with a pH value at its pI its net charge is zero and will not bind to the column. 3. When a protein is in a solution with a pH value below its pI its net charge is positive and binding charge will be negative cation exchange. 4. Small effects: a. Van der Waals b. Non-polar interactions. Samples and Sample Buffer 1. Samples should...

Factors Effecting Selectivity (cont.) Selectivity Good selectivity is equal to the degree of separation between peaks. This is important in determining resolution and depends on: 1. Nature of functional groups 2. Number of functional groups 3. Experimental conditions: a. Ionic strength Elution conditions Selectivity of pH Optimum selectivity equals maximum separation between titration curves of individual proteins (i.e. net charge difference). The order in which proteins are eluted can not always be predicted with absolute certainty since a titration curve reflects the total net charge of a protein....

Components of Ion Exchange Media Matrix 1. High porosity a. Large surface area covered by charged groups b. Advantage with large molecules c. Non-porus are preferable for high resolution separation when diffusion effect must be avoided. 2. Inert matrix a. Immunizes non-specific binding 3. High physical stability a. Insures stable constant packing i. No charge do to extreme changes in ionic strength ii. No change do to pH 4. High physical stability and uniformity of particle size a. High flow rate b. Improved throughput 5. High chemical stability a. Cleaning using stringent solutions. 6. Ion Exchange...

Components of Ion Exchange Media (cont.) Weak and strong ion exchanger refers to the extent that the ionization state of the functional groups varies with pH. Strong ion exchange shows no variation in ion exchange capacity with changes in pH. These exchanges show no variation with pH changes so have no buffering capacity and remain fully charged over a broad pH range. Advantages of Working with Strong Ion Exchange Media 1. Development and optimization separation are fast since charge characteristics do not change or are less variable than weak ion exchange media. 2. The sample interaction is...

Experimental Conditions & Method Optimization Experimental Conditions of Ion Exchange Chromatography Final resolution 1. Particle size 2. Porosity 3. Column packing Separation influence 1. Net surface charge of a protein at a pH 2. The pH of the buffer 3. Ionic strength of the buffer 4. Elution conditions. Capture 1. Intermediate purification 2. Final purification Sample preparation 1. The buffer of the sample should be the same as the starting buffer. 2. For small sample volume diluting the sample in the start buffer in order to lower ionic strength and adjust pH 3. If the pI of the protein...

Experimental Conditions & Method Optimization (cont.) Method Optimization (cont.) pH and Ionic Strength The pH should be as close to the point of release as possible. Note: If the pH is to low or high elution becomes more difficult and high salt concentrations are needed. Note: Avoid extreme changes in pH or other conditions that may cause inactivation or precipitation. Proteins will begin to dissociate from the ion exchange column media at .˜0.5 pH units from there pI, at an ionic strength of ˜0.1M. The pH of the starting buffer should be at least 0.5-1 pH units below the pI of the protein when...